哈密沙虎体内蜥蜴利什曼原虫前鞭毛体的超微结构观察

从内蒙古额济纳旗从哈密沙虎肝组织内培养分离的蜥蜴利什曼原虫前鞭毛体进行了超微结构观察。前鞭毛体多为柳叶状，其膜下微管数平均为５７±８，微管直径为２４ｎｍ，微管间距为１８－２２ｎｍ，质膜厚度为７－８ｎｍ。该原虫的膜下微管数和微管间距均较Ｌｅｉｓｈｍａｎｉａ ａｇａｍａｅ主Ｌ．ａｄｌｅｒｉ两种蜥蜴利什曼原虫为少和窄。     

杜氏利什曼原虫前鞭毛体和无鞭毛体的比较蛋白质组学分析

目的 应用蛋白质组学技术分析杜氏利什曼原虫前鞭毛体和无鞭毛体蛋白质表达状况。 方法 分别提取和纯化杜氏利什曼原虫四川SC6株前鞭毛体与纯培养无鞭毛体的总蛋白,分别经pH3～10的预制胶条进行双向电泳分离,凝胶用考马斯亮蓝染色,凝胶图像以PDQuest 1.0软件分析,并对主要差异表达蛋白点用电喷雾质谱法进行鉴定。 结果 等量的前鞭毛体与纯培养的无鞭毛体总蛋白经双向电泳分离后均可获近700个蛋白点,其中超过90%的蛋白点的分布和相对强度基本一致。与前鞭毛体比较,6个蛋白点在无鞭毛体蛋白中明显高表达,3个蛋白点低表达。6个明显高表达的蛋白点中有5个为已知功能蛋白,分别为Reiske铁硫蛋白前体、α微管蛋白、过氧化物酶1、二氢硫辛酰胺乙酰转移酶前体和甘露糖-1-磷酸瓜氨酸转移酶;3个低表达的蛋白点中有2个为已知功能蛋白,分别为热休克蛋白70和β微管蛋白。这些差异调节表达蛋白与碳水化合物/能量代谢,应激反应,细胞膜和细胞骨架形成相关。 结论 前鞭毛体与无鞭毛体蛋白质的表达存在差异。     

几种利什曼原虫前鞭毛体在不同培养基上生长繁殖的观察

在三恩氏培养基和经过改良的RPMI1640培养基上比较了5株杜氏利什曼,1株沙鼠利什曼和1株蜥蜴利什曼前鞭毛体的繁殖动态。结果表明有四种类型。其中761株杜氏利什曼和蜥蜴利什曼属于一组;杜氏利什曼801株和852株为一组;771株杜氏利什曼与沙鼠利什曼为一组,自1株杜氏利什曼本身为一组。7株利什曼在两种培养基上处于繁殖峰值的原虫数分别为2.8～3.8×10~7和3.9～7.9×10~6。讨论了这四种繁殖动态类型的生物学意义,并认为经过补充的RPMI 1640培养基可供在利什曼原虫的生物化学和免疫学研究中应用。     

不同利什曼原虫分离株在培养基中生长繁殖观察

目的　比较我国不同类型内脏利什曼病流行区利什曼原虫前鞭毛体在不同培养基中的生长繁殖情况，为选择合适培养基用于利什曼原虫培养提供实验依据。方法　将3 ×105个KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体分别接种至1 mL NNN培养基、1 mL M199 + 20%胎牛血清培养基、1 mL M199 + 20%马血清培养基及1 mL 脑心浸液培养基（含血红素）中，22 ℃温箱中无菌静置培养，显微镜下连续观察计数8 d，绘制3株利什曼原虫前鞭毛体的生长曲线。 结果 KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均能在NNN培养基、M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基中生长繁殖，在NNN培养基中培养不同时间后的3株利什曼原虫前鞭毛体计数均显著高于M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基（P均 < 0.05），在这3种培养基中培养不同时间后的KS?2株利什曼原虫前鞭毛体计数均显著高于Cy和JIASHI?5株（P均 < 0.05）。KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均不能在脑心浸液培养基中生长繁殖。结论　分离自我国不同类型内脏利什曼病流行区的利什曼原虫在同一培养基中生长增殖速度有差异，同一利什曼原虫分离株在不同培养基中的生长繁殖速度亦有差异。NNN培养基是最适合我国内脏利什曼病流行区利什曼原虫分离株的培养基。     

不同利什曼原虫分离株在培养基中生长繁殖观察

目的　比较我国不同类型内脏利什曼病流行区利什曼原虫前鞭毛体在不同培养基中的生长繁殖情况,为选择合适培养基用于利什曼原虫培养提供实验依据。方法　将3 ×105个KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体分别接种至1 mL NNN培养基、1 mL M199 + 20%胎牛血清培养基、1 mL M199 + 20%马血清培养基及1 mL 脑心浸液培养基（含血红素）中,22 ℃温箱中无菌静置培养,显微镜下连续观察计数8 d,绘制3株利什曼原虫前鞭毛体的生长曲线。 结果 KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均能在NNN培养基、M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基中生长繁殖,在NNN培养基中培养不同时间后的3株利什曼原虫前鞭毛体计数均显著高于M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基（P均 < 0.05）,在这3种培养基中培养不同时间后的KS?2株利什曼原虫前鞭毛体计数均显著高于Cy和JIASHI?5株（P均 < 0.05）。KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均不能在脑心浸液培养基中生长繁殖。结论　分离自我国不同类型内脏利什曼病流行区的利什曼原虫在同一培养基中生长增殖速度有差异,同一利什曼原虫分离株在不同培养基中的生长繁殖速度亦有差异。NNN培养基是最适合我国内脏利什曼病流行区利什曼原虫分离株的培养基。     

酸碱度对不同来源杜氏利仁曼原虫前鞭毛体生长情况的影响

酸碱度对杜氏利什曼原虫鞭毛体（以下简称鞭毛体）生长情况的影响,前人做过许多实验,但对不同来源鞭毛体生长情况的对比观查,尚未见报道。本文阐述了酸碱度对不同来源鞭毛体生长情况的影响。值得注意的是在pH值4.6,8.6时,鞭毛体的生长受到抑制,以球型形态多见。鞭毛体是利什曼原虫的体外培养形态,了解酸碱度对鞭毛体生长情况的影响,尤其是对抗锑种株生长情况的影响,进一步了解鞭毛体在体外培养时的生长特征及各种株间的生长差异,为黑热病的防治提供一些基础资料。     

克拉玛依地区的利什曼病Ⅶ.大沙鼠体内利什曼原虫前鞭毛体的超微结构观察

在新疆克拉玛依小拐农场,从大沙鼠耳组织内查见的利什曼原虫,经NNN基培养11天,对原虫的前鞭毛体作超微结构观察。前鞭毛体多为柳叶状。其膜下微管数平均为80±9(68～111)个,微管直径为24nm,间距为15～28nm,质膜厚度为8nm,与吴传芬等和王捷等报道的甘肃沙鼠利什曼原虫的前鞭毛体有明显差别;在胞质内广泛分布着内质网,线粒体发达,高尔基体常见于线粒体一动基体复合体附近。在鞭毛基体上部有基板1和基板2。在胞质内还可看到脂滴和空泡。     

Requirements of defined cultivation conditions for standard growth of Leishmania promastigotes in vitro

The growth characteristics of L. chagasi (MHOM/BR/79/LI01) and L. braziliensis (MHOM/BR/72/1670), the causative agents of visceral and muco-cutaneous leishmaniases, respectively, were compared. Inoculum size clearly influences the growth course of both Leishmania species, whatever the culture medium used (serum-supplemented media: GLSH or RPMI, and a chemically defined medium: LITR9). Cultures initiated with low concentrations failed to promote cell growth, while typical growth curves were obtained when higher promastigote inocula were used. For all the species tested, the higher the initial density of flagellates in the medium, the shorter were the periods covered by the latent and particularly by the logarithmic growth phases. In contrast, using constant inocula, variations in the volume of the incubation medium did not change the time-course of the different culture phases of either Leishmania species, provided that the ratio of incubation medium to total flask volume was comparable. Only cell division time significantly increased with the culture volume. We also determined whether or not the growth characteristics of the promastigotes of L. chagasi or L. braziliensis could be generalized to other members of the genus. Our results show that, whatever the culture medium used, L. infantum behaves in the same way as does L. chagasi, whereas L. panamensis, L. guyanensis, L. mexicana and L. amazonensis display growth patterns similar to that of L. braziliensis.     

Effect of ethidium bromide on growth and morphology of Leishmania tarentolae promastigotes in vitro.

Incubation of Leishmania tarentolae promastigotes in 0.01 microgram/ml ethidium bromide in Neo Ye medium for 96 h resulted in 60% inhibition of cell growth and 91% dyskinetoplasty. After 48 h incubation in ethidium bromide over 50% of the cells were scored dyskinetoplastic by light microscopy although the electron microscopical examination revealed that most promastigotes contained at least a small amount of kDNA. A few of the treated cells undergoing division contained two kinetoplasts--one devoid of kDNA and the other with a reduced amount of kDNA as seen in the electron micrographs.     

The interaction of Leishmania donovani promastigotes and human fibroblasts in vitro

Leishmania donovani promastigotes derived from infected hamster spleens, in either log phase or stationary phase growth, associated with human foreskin fibroblasts in vitro and assumed the morphological characteristics of amastigotes. This apparent conversion was noted within hours at 26 degrees C, 32 degrees C or 37 degrees C; in the continued presence of promastigotes, increasing numbers of amastigote-like forms were seen for 2 weeks at 26 degrees C or 32 degrees C. At 37 degrees C amastigote-like forms declined sharply after 6 days. Multiplication of amastigote-like forms was not observed at any temperature, this was also true of freshly isolated amastigotes from hamster spleens which associated with fibroblasts but did not multiply. Approximately 0.1% of promastigotes appeared to convert per day. Amastigote-like forms were seen within fibroblasts by transmission electron microscopy, surrounded by a closely applied host membrane. Scanning electron microscopy showed promastigotes with their flagellae under or within fibroblasts, but phagocytosis was not observed. These experiments suggest that the conditions required for promastigote-to-amastigote conversion may be different than those required for amastigote multiplication, and the mammalian core body temperature may not be required for promastigote conversion.     

Effects of long-term in vitro cultivation on the virulence of cloned lines of Leishmania major promastigotes.

The virulences of several clones from a single Leishmania major strain were studied in BALB/c mice. Clones showed the same pattern of infectivity and virulence two months after cloning as the parental population. After prolonged in vitro culture, however, it was apparent that two types of virulent clones existed: although the level of virulence remained stable in some clones, in others, such as C-11, it progressively decreased, as in the parental population. The progressive loss in virulence in a continuously cultured mixed population was probably due to selection, as the initial mixture of a stable virulent clone and a stable avirulent clone eventually yielded a totally avirulent promastigote population. After cultivation for 12 months, neither clone C-11 nor the parental population produced lesions in inoculated mice but virulent parasites were recovered from the inguinal nodes of the mice, possibly as a result of selection in vivo for virulent parasites.     

Insulin-like growth factor I is a growth-promoting factor for Leishmania promastigotes and amastigotes

Leishmaniases are diseases caused by protozoa of the genus Leishmania that affect more than 20 million people in the world. The initial phase of the infection is fundamental for either the progression or control of the disease. The Leishmania parasites are injected in the skin as promastigotes and then, after been phagocytized by the host macrophages, rapidly transform into amastigotes. In this phase different nonspecific cellular and humoral elements participate. We have shown previously that insulin-like growth factor (IGF)-I that is constitutively present in the skin induces growth of Leishmania promastigotes. In the present paper we show further evidence for the importance of this factor: (i) IGF-I also can induce a growth response in Leishmania (Leishmania) mexicana amastigotes; (ii) IGF-I binds specifically to a putative single-site receptor on both promastigotes and amastigotes; (iii) IGF-I induces a rapid tyrosine phosphorylation of parasite proteins with different molecular mass in promastigotes and amastigotes of L. (L.) mexicana; and, finally, (iv) the cutaneous lesion in the mice when challenged by IGF-I-preactivated Leishmania (Viannia) panamensis is increased significantly because of inflammatory process and growth of parasites. We thus suggest that IGF-I is another important host factor participating in the Leishmania–host interplay in the early stage during the establishment of the infection and presumably also in the later stages.     

Preservation of Leishmania donovani promastigotes in blood agar slants

Long-term cultivation of Leishmania promastigotes by weekly passage to fresh medium was reported to be disadvantageous because needs labor, risk of contamination, lowering in infectivity and virulence pattern. Cryopreservation and Lyophilization require expensive facilities which could be a burden and unaffordable to most laboratories of developing countries where the disease is endemic. These problems could be minimized by simple preservation of Leishmania donovani promastigotes in blood agar slants at 7-8 degrees C for 6-7 months. The preserved promastigotes were examined for viability up to one year at a regular interval of one month. Viable promastigotes were found and revived successfully from all the slants stored up to 7 months after that, the viability of promastigotes was found to be decreased in the slants of 8-9 month storage. No viable promastigotes were recovered from the slants stored up to 11-12 months. By this method, the promastigotes can easily be stored up to 7 months without loss of biological activity. The number of passage of promastigotes to fresh medium has been greatly reduced by this method from 30 times to 01 when compared with weekly passage in liquid medium. This simple and economical method can be recommended for short storage of Leishmania culture without loss of any activity.     

pH homeostasis in Leishmania donovani amastigotes and promastigotes.

Intracellular pH and pH gradients of Leishmania donovani amastigotes and promastigotes were determined over a broad range of extracellular pH values. Intracellular pH was determined by 31P NMR and by equilibrium distribution studies with 5,5-dimethyloxazolidine-2,4-dione or methylamine. Promastigotes maintain intracellular pH values close to neutral between extracellular pH values of 5.0 and 7.4. Amastigote intracellular pH is maintained close to neutral at external pH values as low as 4.0. Both life stages maintain a positive pH gradient to an extracellular pH of 7.4, which is important for active transport of substrates. Treatment with ionophores, such as nigericin and carbonyl cyanide m-chlorophenylhydrazone and the ATPase inhibitor dicyclohexylcarbodiimide, reduced pH gradients in both stages. Maintenance of intracellular pH in the physiologic range is especially relevant for the survival of the amastigote in its acidic in vivo environment.